This projects attempts to create the dentin-pulp complex in-vitro using microfabrication and dental pulp stem cells (DPSC). The dentin pulp interface is characterized by a lining of specialized cells of unique morphology that produce the collagenous dentin matrix and control its mineralization. Highly polarized odontoblasts with diameters of 5 to 10 m are tightly packed along the pulpal wall. Their unique feature is the odontoblastic process that extends up to 10 mm deep along tubules into the mineralized dentin. The cells are anchored in this position for the lifetime of the tooth. Hence these cells can live fr decades and only respond to external insult by producing additional dentin matrix (reparative dentin). The surrounding dentin pulp contains progenitor cells that can differentiate into odontoblast cells and produce dentin matrix or form a dentin crown when implanted with epithelial or ameloblast-like stem cells into an immunocompromised mouse. This application proposes to reconstitute this highly organized arrangement of cells in-vitro using tissue engineering approaches. In collaboration with the bioengineering laboratory of Dr. Tejal Desai and the microfabrication facilities at the University of California at Berkeley, micropatterned polymer scaffolds will be generated that permit the positioning of cells in precise locations. In our design these locations will be aligned with an open cylindrical pore allowing for one cell being positioned on top of each pore. Chemotactic approaches will be used to encourage protrusion of odontoblastic processes into the pores resulting in a cellular configuration of polarized cells that is comparable to the dentin-pulp interface. These studies will produce a unique construct to investigate gene expression and cellular interactions in-vitro and improve our understanding of tooth development. Furthermore such pre-assembled mimicries of dental tissue will advance current approaches towards tooth regeneration and pulp revitalization. PUBLIC HEALTH RELEVANCE: This project proposes to fabricate micropatterned membranes that will position dental pulp stem cells in-vitro in a configuration similar to the dentin-pulp interface in human teeth. The cell tissue engineered construct will facilitate future molecular biology studies on epithelium/mesenchyme interactions as well further enhance approaches towards engineering of dental tissues.